Introduction

Resistance-size cerebral arteries control brain regional blood flow and maintain perfusion during changes in arterial pressure. One important functional stimulus that controls cerebral artery contractility is intravascular pressure. An elevation in intravascular pressure stimulates depolarization, leading to the activation of smooth muscle cell voltage-dependent calcium (Ca2+) channels, an intracellular calcium concentration ([Ca2+]i) elevation, and vasoconstriction.1 This “myogenic response” regulates regional brain blood flow, maintains perfusion over a range of intravascular pressures, and provides a baseline diameter from which other stimuli can either dilate or constrict. Several pathologies, including hypertension, are associated with altered myogenic responsiveness.2 Therefore, defining mechanisms that control the myogenic response is critical to a better understanding of vascular diseases.

Arterial smooth muscle cell cation channels, including CaV1.2, several K+ and nonselective transient receptor potential (TRP) channels, control vascular contractility.1,2 Multiple TRP channels also contribute to pressure-induced depolarization, leading to vasoconstriction, although mechanisms involved are unclear.2 In contrast, vascular contractility regulation by arterial smooth muscle cell anion channels is poorly understood. Chloride (Cl−) is the most abundant intracellular anion in vascular smooth muscle cells, with intracellular [Cl−] ≈50 mmol/L.3 The estimated reversal potential (Erev) for Cl− in smooth muscle cells is between −30 and −20 mV.4 The entire working range of rat cerebral arteries, from fully dilated to fully constricted, occurs between membrane potentials of ≈−60 and −20 mV, which elevates global arterial wall [Ca2+]i from ≈100 to 350 nmol/L.5 With physiological ionic gradients, Cl− channel activation would result in Cl− efflux and arterial myocyte depolarization and vasoconstriction.1 This is in contrast to some other cell types, including adult neurons, where Cl− Erev is ≈−75 mV, a voltage near resting potential.6 The concept that Cl− channels contribute to myogenic constriction has previously been suggested from experiments that used highly nonspecific pharmacological Cl− channel modulators.1,3,7,8 Indeed, poor selectivity of pharmacological Cl− channel modulators and uncertain molecular identity of the protein(s) involved has hindered progress in defining functions of Cl− channels in contractile arterial smooth muscle cell and their involvement in the regulation of vascular contractility.

Transmembrane protein 16A (TMEM16A) channels, also termed Anoctamin 1, are recently discovered Ca2+-activated Cl− (ClCa) channels.9–11 Our group and others recently demonstrated that TMEM16A channels are expressed in arterial smooth muscle cells and generate ClCa currents.12–15 TMEM16A has recently been described as a negative regulator of arterial smooth muscle cell proliferation.15 However, regulation of contractility by arterial smooth muscle cell TMEM16A channels is unclear. In the present study, we demonstrate that cell swelling and pressure-induced membrane stretch stimulate TMEM16A channels in arterial smooth muscle cells, leading to depolarization and vasoconstriction. Data also suggest that membrane distention activates nonselective cation channels that stimulate TMEM16A channels through local Ca2+ signaling. Thus, we show that arterial smooth muscle cell TMEM16A channels are one component of a mechanosensitive mechanism that contributes to the myogenic response.

TMEM16A Channel Knockdown

Three small interference RNA (siRNA) sequences targeting TMEM16A or negative control siRNA (Invitrogen), as used previously,12 were inserted intracellularly into cerebral arteries using either reverse permeabilization, as described,17–21 or a Bex CUY21Vivo-SQ electroporator. Arteries were then maintained in serum-free DMEM F12 media supplemented with 1% penicillin-streptomycin (Sigma) for 4 days after reverse permeabilization or 3 days after electroporation at 37°C in a sterile incubator (21% O2, 5% CO2). Western blotting was used to compare the effect of TMEM16A siRNA with control siRNA on protein expression. Band intensity of proteins from arteries treated with either TMEM16A siRNA or control siRNA were compared on the same membranes. Reverse permeabilization and electroporation similarly reduced TMEM16A protein (reverse permeabilization, 62 ± 5% of control siRNA, n=7; electroporator, 56 ± 1% of control siRNA, n=3) in arteries (P>0.05).

Membrane potential measurements were obtained in arteries at 10 or 60 mm Hg that had developed steady-state myogenic tone. This was done by maintaining arteries at steady pressure for at least 2hours and confirmed using edge detection. Membrane potential was measured by inserting glass microelectrodes filled with 3 mol/L KCl (50–90 mΩ) into the adventitial side of pressurized arteries. Membrane potential was recorded using a WPI FD223 amplifier and digitized using pClamp 9.2 software (Axon Instruments) and a personal computer. Criteria for successful intracellular impalements were (1) a sharp negative change in potential on insertion; (2) stable voltage for at least 1 minute after entry; (3) a sharp positive voltage deflection on exit from the recorded cell; and (4) a <10% change in tip resistance after the impalement.

Statistical Analysis

OriginLab and GraphPad InStat software were used for statistical analyses. Values are expressed as mean±SEM. Student t test was used for comparing paired and unpaired data from 2 populations, and ANOVA with Student-Newman-Keuls post hoc test was used for multiple group comparisons. P<0.05 was considered significant. Power analysis was performed on all data in which P>0.05 to verify that sample size was sufficient to give a power value of >0.8. All-points histograms were fit with a multipeak gaussian function, using Microcal Origin.

Expanded materials and methods are provided in the Online Data Supplement.

Pressure-induced membrane stretch activates TMEM16A channels in arterial smooth muscle cells. A, Reduction in pipette pressure from 0 to −40 mm Hg stimulates inward currents that are partially inhibited by the TMEM16A-inhibitory antibody. Traces represent the average of current recordings of 29 control and 18 TMEM16A antibody-exposed patches at −80 mV. B, Exemplary recordings of single channels activated by −40 mm Hg pressure and inhibited by the TMEM16A-inhibitory antibody. C, All-points histograms of arterial smooth muscle cell patches fit with a gaussian function for −40 mm Hg pressure in the absence and presence of the TMEM16A antibody. D, Original cell-attached recordings of recombinant TMEM16A channels expressed in HEK293 cells in the absence and presence of the TMEM16A inhibitory antibody. No patch pressure was applied during experiments on HEK293 cells. E, All-points histograms of recombinant TMEM16A channels fit with a gaussian function in the absence and presence of the TMEM16A antibody.

Discussion

The regulation of vascular contractility by anion channels is poorly understood. Similarly unclear are physiological functions and mechanisms of regulation of TMEM16A channels in contractile arterial smooth muscle cells. In the present study, we show that TMEM16A channels control smooth muscle cell membrane potential and contractility and contribute to the myogenic response in cerebral arteries. We show that cell swelling activates TMEM16A currents and pressure-induced membrane stretch activates single TMEM16A channels in arterial smooth muscle cells. Our data also indicate that nonselective cation channels generate a local intracellular Ca2+ signal that activates TMEM16A currents. These data provide a mechanism by which pressure-induced activation of arterial myocyte nonselective cation channels stimulates TMEM16A currents, leading to arterial smooth muscle cell depolarization and vasoconstriction.

Two distinct types of ClCa currents are present in vascular myocytes: “classic” ClCa and cGMP-dependent ClCa currents.25,26 ClCa currents have been characterized in myocytes of several vascular beds as outwardly rectifying Cl− currents that are activated by [Ca2+]i.3,27 Outward rectification of classic ClCa currents at nanomolar [Ca2+]i is linearized by an elevation in [Ca2+]i.27 Cell swelling activates Cl− currents in cerebral, pulmonary, and renal artery and portal vein smooth muscle cells.8,23,24 A reduction in extracellular Cl− elevated myogenic tone and nonselective Cl− channel blockers hyperpolarized and dilated pressurized cerebral arteries.7 Cl− efflux, measured using self-referencing ion-selective electrodes, also correlated with the myogenic response.28 Our data obtained using extracellular ionic replacement, inhibitory antibodies, RNAi, and comparison of single channel properties to recombinant TMEM16A channels indicate that cell swelling, pressure-induced membrane stretch, and intravascular pressure activate TMEM16A channels in arterial smooth muscle cells. We show that linearization of the I-V relationship by cell swelling and the relative permeability of swelling-activated currents to I− and Cl− is also similar to that of recombinant TMEM16A channels.9 We also demonstrate that selective TMEM16A knockdown attenuates intravascular pressure–induced arterial depolarization and vasoconstriction. These data indicate that smooth muscle cell TMEM16A channels contribute to myogenic constriction in cerebral arteries. Recent studies demonstrated that TMEM16A channels are expressed in smooth muscle cells of rat small cerebral and mouse conduit arteries, cultured rat pulmonary artery smooth muscle cells, and interstitial cells of Cajal.12–15,29 Another recent study demonstrated that T16Ainh-A01, a TMEM16A current inhibitor with unclear selectivity, reduced a chronic hypoxia-induced elevation in serotonin contraction in rat pulmonary arteries.30 These findings suggest that smooth muscle cell TMEM16A channels may regulate contractility not only in cerebral arteries but in anatomically diverse vasculature and other smooth muscle cell types.

We show that removal of extracellular Ca2+ and replacement of intracellular EGTA with BAPTA abolished swelling-activated TMEM16A currents. These data are similar to those from a previous study that measured Cl− current regulation by cell swelling in smooth muscle cells of the basilar artery, a large cerebral vessel.24 In contrast, swelling-induced TMEM16A currents were not altered by thapsigargin or nimodipine, arguing against the functional involvement of SR Ca2+ release and voltage-dependent Ca2+ channels. A previous report described that swelling activated nonselective cation currents but did not stimulate Cl− currents in cerebral artery smooth muscle cells.22 In this earlier study, intracellular and extracellular solutions were Ca2+-free. Therefore, these data are consistent with ours that swelling-induced Ca2+ influx activates TMEM16A currents. To determine the mechanism by which cell swelling activates TMEM16A channels, we tested the hypothesis that nonselective cation channels, which have been previously implicated in mediating myogenic constriction, were involved. This approach also permitted us to test the associated hypothesis that TMEM16A channels may be mechanosensitive. Our data show that Gd3+ and SKF96365 blocked swelling-activated TMEM16A currents but did not alter currents generated by recombinant TMEM16A channels in HEK293 cells. These data indicate that swelling activates nonselective cation channels, leading to Ca2+ influx that stimulates TMEM16A channels. Consistent with our data, Gd3+ blocked both swelling- and pressure-induced depolarization in cerebral artery smooth muscle cells.22 In contrast, swelling-activated Cl− currents dissimilar to classic ClCa were not inhibited by BAPTA in portal vein smooth muscle cells.23 Depolarization-induced Cl− currents attributed to TMEM16A have been described in interstitial cells of Cajal.29 Based in part on their activation latency after stimulation, these Cl− currents have been suggested to be activated by Ca2+-induced Ca2+ release.29 Collectively, these studies suggest that diverse mechanisms of ClCa current activation may exist in smooth muscle cells of different tissues. Future studies will determine if these different activation mechanisms also apply to regulation of TMEM16A channels. Our data indicate that a mechansosensitive mechanism stimulates nonselective cation channels that activate TMEM16A via local Ca2+ signaling in cerebral artery smooth muscle cells. These data also suggest that arterial smooth muscle cell TMEM16A currents are not directly activated by cell swelling.

The molecular identity of nonselective cation channels that stimulate TMEM16A currents was not determined in the present study. We show that membrane stretch induced by negative pipette pressure stimulates single TMEM16A channels in arterial smooth muscle cells. In a majority (≈80%) of membrane patches, pressure activated currents to which multiple simultaneously gating channels contributed. The TMEM16A-inhibitory antibody reduced stretch-activated currents by ≈50%, indicating that TMEM16A channels contribute almost half of the current. In a minority of patches (≈20%), single ion channels identical to recombinant TMEM16A channels were activated. These data suggest that TMEM16A channels may cluster in the plasma membrane with other stretch-activated channels, consistent with other evidence in this study that closely localized nonselective cation channels activate TMEM16A after membrane stretch. Several nonselective cation channels expressed in arterial smooth muscle cells are Ca2+-permeant, including multiple TRPC and TRPM subfamily members, TRPP1/2, TRPV2, and TRPV4.2 Conceivably, 1 or more of these channels, including TRP heteromultimers, may control TMEM16A channel activity. Previous studies have indicated that TRPC6, TRPM4, and TRPP1/2 activation contributes to the myogenic response.2,31 Under physiological conditions, TRPC6 and TRPP1/2 activation would lead to both Na+ and Ca2+ influx.32 In contrast, TRPM4 channels are primarily Na+-permeant.32 Our data support the concept that both local Ca2+ signaling and Na+ influx mediated by nonselective cation channels contribute to pressure-induced depolarization and vasoconstriction.2 Data indicate that cell swelling did not stimulate TMEM16A currents via activation of voltage-dependent Ca2+ channels or intracellular Ca2+ release. This finding is consistent with evidence that voltage-dependent Ca2+ channel activation does not contribute to pressure-induced depolarization, but produces the depolarization-induced global [Ca2+]i elevation, and that acute Ca2+ store depletion inhibits Ca2+ sparks, leading to vasoconstriction.1 Given the large number of potential candidates, that currently unidentified channels may be involved, more than 1 channel may be engaged, and heteromultimeric proteins may be involved, it was beyond the scope of this study to determine the molecular identity of nonselective cation channels that activate TMEM16A channels. Future studies should be designed to identify Ca2+-permeant ion channels that control TMEM16A channels in arterial smooth muscle cells.

The contribution of TMEM16A channels to myogenic vasoconstriction was studied between 20 and 100 mm Hg, a range that encompasses physiological intravascular pressures in the cerebral circulation. Physiological cerebral artery membrane potential over this range of pressures is ≈−65 to −36 mV.5,33 The predicted Erev for Cl− is ≈−30 mV, indicating that TMEM16A channel–mediated Cl− efflux would contribute to membrane depolarization and myogenic constriction over the range of pressures studied. Elevating pressure above 100 mm Hg does not further depolarize cerebral arteries with a plateau at ≈−30 mV, a potential similar to the predicted Cl− Erev.5,33 Data in the present study indicate that the RNAi-mediated reduction in TMEM16A protein (≈43%) and myogenic response (43% to 49%) was similar over the entire pressure range. These data suggest that TMEM16A channels contribute equally to myogenic constriction over this pressure range. These observations could be interpreted as indicating that TMEM16A channels are the major contributor to pressure-induced depolarization and that the Cl− reversal potential determines maximal depolarization. However, multiple mechanisms can contribute to the pressure-induced depolarization plateau. At voltages more positive than ≈−30 mV, Cl− efflux will switch polarity to influx that will oppose depolarization mediated by nonselective cation current. Pressure-induced depolarization is also opposed through the activation of K+ channels, including Kv and BKCa.1 Although the RNAi-mediated reduction in TMEM16A protein and myogenic response were similar, the reduction in swelling-activated TMEM16A currents was larger. Explanations for this result include that a threshold level of TMEM16A protein may be required for the formation of functional ion channels in arterial smooth muscle cells. In addition, multiple processes contribute to pressure-induced depolarization and myogenic constriction, with some of these mechanisms interacting, as we show in the present study.2 Therefore, the partial loss of 1 signaling component may lead to amplification of functional effects. Future studies should examine the relative contribution of nonselective cation and TMEM16A channels to the myogenic vasoconstriction both in vitro and in vivo. This determination would require the molecular identification of the nonselective cation channels that communicate with TMEM16A.

Arterial smooth muscle cell chloride (Cl−) channels may also regulate vascular contractility and contribute to the myogenic response, but defining physiological functions of these channels in the vasculature has been hindered by uncertain identity of protein(s) involved and a lack of selective modulators.

The myogenic response is a physiological smooth muscleenspecific mechanism that controls systemic blood pressure and regional organ blood flow. Cardiovascular diseases, including hypertension, are associated with an augmented myogenic response, which elevates blood pressure and can induce end-organ damage. Intravascular pressure has been proposed to activate several different nonselective cation channels, including members of the TRP family, to induce myogenic vasoconstriction. The concept that Cl− channels regulate vascular contractility has been suggested from experiments that used Cl− channel modulators with low or uncertain specificity. However, the molecular identity of Cl− channels that control vascular contractility and contribute to the myogenic response was unclear. In the present study, we used a combination of molecular, electrophysiological, and functional approaches to show that membrane stretch activates TMEM16A channels in cerebral artery smooth muscle cells. Our data suggest that a stretch-induced local intracellular Ca2+ signal generated by nonselective cation channels stimulates TMEM16A channels. Intravascular pressure–induced TMEM16A channel activation contributes to membrane depolarization and vasoconstriction. These data indicate that TMEM16A channels are one component of a mechanosensitive mechanism that contributes to the myogenic response. These results also identify a new approach to modulate the myogenic response through the manipulation of TMEM16A channel activity.